Abstract

Given the prevalence of Ras mutations in human cancer, it is critical to understand the effector pathways downstream of oncogenic Ras leading to transformation. To directly assess the requirement for Rac1 in K-ras–induced tumorigenesis, we employed a model of lung cancer in which an oncogenic allele of K-ras could be activated by Cre-mediated recombination in the presence or absence of conditional deletion of Rac1. We show that Rac1 function is required for tumorigenesis in this model. Furthermore, although Rac1 deletion alone was compatible with cell viability and proliferation, when combined with K-ras activation in primary epithelial cells, loss of Rac1 caused a profound reduction in proliferation. These data show a specific requirement for Rac1 function in cells expressing oncogenic K-ras. [Cancer Res 2007;67(17):8089–94]

Rac1

K-ras

lung cancer

tumorigenesis

Cre-mediated recombination

Introduction

The Rac proteins are members of the Rho family of small G proteins and are implicated in the regulation of several pathways, including those leading to cytoskeleton reorganization, gene expression, cell proliferation, and survival. The deregulation of these pathways is a reoccurring theme in transformation and tumorigenesis; however, the role of the Rac family proteins in cancer has not been fully elucidated. Previous reports have indicated that activated mutated alleles of Rac and cdc42 can be transforming when ectopically expressed in cells (
1,
2). However, tumor-associated mutations in the Rho family genes have not been reported (reviewed by ref.
3). Some reports suggest that the overall activity of Rac1 and cdc42 seems to be up-regulated in some forms of cancer, and this has been suggested to be important for tumor initiation and progression (
4–
6). Additional evidence for a role for Rac proteins in transformation arises from the involvement of Rho family members in Ras-induced transformation. Initial studies in vitro showed that dominant-negative forms of Rac1 and cdc42 can inhibit the transformation of cells by ectopically expressed alleles of activated Ras (
7,
8). In addition, genetic studies have shown that Rac2 is required for Ras-induced proliferation of Mast cells in mice which have lost the Ras-GAP Nf1 (
9). Deletion of the Rac regulator Tiam1 led to decreased levels of active Rac1, 2, and 3 and delayed skin tumor initiation and progression induced by mutations in the H-Ras allele in response to 7,12-dimethylbenzanthracene (DMBA) treatment (
10).

Previous studies have shown that Ras signaling can activate Rac through both phosphoinositide-3-kinase (PI3K)–dependent and PI3K-independent mechanisms (
11,
12). However, other studies have shown that Ras activation can down-regulate levels of activated Rac (
13,
14). Thus, although Ras affects the levels of Rac-GTP, the nature of this regulation is not clear because previous results have been contradictory. It is possible that the differences observed in previous studies are due to tumor/cell type differences and/or differences in the cellular environment (reviewed by ref.
3).

To directly assess the requirement for Rac1 in K-ras–induced tumorigenesis, we employed a conditional model of lung cancer in which an oncogenic allele of K-ras is activated by Cre-mediated recombination. By combining conditional activation of K-ras with conditional deletion of Rac1 (
15), we show that Rac1 is required for tumorigenesis in this system.

Materials and Methods

Generation of Rac1flox/flox and LSL-K-rasG12D mice and lung infections with adenovirus. As previously described (
15,
16).

Isolation and infection of primary epithelial kidney (BMK) cells. Baby mouse kidney (BMK) cells from the various genotypes were isolated as described previously (
17) and plated at 80% confluence in 10-cm dishes (for cell cycle analysis) or at 2 × 104 cells per well in 24-well dishes (for cell counting experiments). BMKs were treated with either 1 × 107 plaque-forming units (PFU) per 10-cm dish of control Ad virus or Ad-Cre for 24 h. After infection, 72 h later, cells were harvested for analysis of propidium iodide (PI) distribution, and cell counts were done 72 and 96 h postinfection. For PI staining, cells were fixed overnight in 70% ethanol. Following washing in PBS, cells were resuspended in sample buffer containing 50 μg/mL propidium iodide and 0.2 mg/mL RNase A and were analyzed within 24 h on a Coulter EPICS XL-MCL. The status of the K-rasG12D allele in the BMKs and tumors was analyzed by PCR as previously described (
15). Analysis of the Rac1 allele in tumors and BMKs was done by PCR using the following primers: P1 = 5′-GTTGAAGGTGCTAGCTTGGGAACTG-3′, P2 = 5′-GAAGGAGAAGAAGCTGACTCCCATC-3′, P3 = 5′-CAGCCACAGGCAATGACAGATGTTC-3′.

Histologic analysis and immunohistochemistry. Animals were sacrificed at the indicated time points. All histologic analysis was done as described (
18). Immunohistochemistry was done on 4-micron paraffin sections. Briefly, endogenous peroxidases were blocked in 3% H2O2 for 10 min at room temperature and then in TBS with 0.1% Triton X-100 and 10% normal goat serum at 37°C for 1 h. Sections were incubated first with a Rac1 antibody (1:500 dilution; Upstate Biotechnology) and then a secondary antibody in TBS with 0.1% Triton X-100 and 10% normal goat serum at 37°C for 30 min. Staining was visualized with the Vectastain ABC kit (Vector).

Results and Discussion

To assess directly whether Rac1 is involved in the initiation and/or progression of K-ras–induced lung tumors, we employed the LSL-K-rasG12D mouse model of lung adenocarcinoma. In this model, lung tumor initiation is achieved by infection with an adenovirus expressing Cre-recombinase (Ad-Cre), which leads to removal of a transcriptional stop element and activation of an oncogenic allele of K-rasG12D under physiologic control. In this model, infected animals develop numerous areas of hyperplasia, followed by adenoma and adenocarcinoma formation (
15). Mice with a conditional loss-of-function allele of Rac1 (Rac1flox/flox; ref.
16) were crossed to the LSL-K-rasG12D animals to generate LSL-K-rasG12D;Rac1flox/flox and control LSL-K-rasG12D;Rac1flox/+ mice. In the former, Cre-mediated recombination should result in the activation of the K-rasG12D allele and simultaneous Rac1 inactivation in infected cells of the lung; the LSL-K-rasG12D;Rac1flox/+ mice would retain a functional Rac1 allele following Cre-mediated recombination. Rac1flox/flox mice were used as an additional control for possible effects associated with the loss of Rac1 function.

Following intranasal administration of Ad-Cre, mice from each group were sacrificed at 6, 12, 18, and 24 weeks postinfection. Both tumor number and volume were assessed by histologic examination. As shown in
Fig. 1
, the LSL-K-rasG12D;Rac1flox/+ mice displayed hyperplasia and adenomas (an average of six lesions per animal) already at the 6-week time point. The number of discernible tumors in this group of animals progressed to an average of 38 per animal at 12 weeks. At 18 and 24 weeks, the tumors were so large and numerous that individual tumors could not be scored (
Figs. 1B and
2
). Tumor progression was similar to that reported previously for the LSL-K-rasG12D mice (
15). Strikingly, the LSL-K-rasG12D;Rac1flox/flox mice had significantly reduced tumor numbers at all time points. At 6 weeks postinfection, little hyperplasia and few tumors were detected in the majority of the animals; on average, they displayed less than one tumor per animal. At the 12-week time point, animals from this group had an average of three tumors per animal, increasing to approximately nine tumors per animal at the 18- and 24-week time points (
Figs. 1B and
2 and not shown). Thus, loss of Rac1 function dramatically affects tumor formation initiated by oncogenic K-ras.

Analysis of lung tumors in LSL-K-rasG12D;Rac1flox/+ and LSL-K-rasG12D;Rac1flox/flox mice. Mice were treated with Ad-Cre (5 × 106 PFU) and sacrificed at the indicated time points; lungs were examined histologically for the presence of tumors. A, average tumor-to-lung volume ratio (T/L) at the different time points. The difference in the T/L is statistically significant at all time points. Bars, SD; P < 0.005; χ2. B, average incidence of hyperplastic lesions and tumors at the 6- and 12-wk time points. The difference in tumor incidence is statistically significant at both time points. Bars, SD; P < 0.001; χ2. Tumors at later time points were not scored because tumors were fused, and discrete tumors were difficult to identify. C, survival rate of mice infected with Ad-Cre. Mice from both genotypes were infected at the same age, and survival was followed over time. Data were presented by Kaplan-Meier plot. D, distribution of tumor sizes at 6, 12, and 18 wks postinfection with Ad-Cre. The differences between the two mouse groups at all time points are statistically significant (Kolmogorov-Smirnov test).

Time course progression of tumors in the LSL-K-rasG12D;Rac1flox/+ and LSL-K-rasG12D;Rac1flox/flox mice. Mice were sacrificed at 6, 12, 18, and 24 wks postinfection. Top and middle, magnification, ×4; bottom, magnification, ×10. Representative histologic findings from both groups at these time points are shown. Arrowheads, regions of hyperplasia; arrows, adenomas. Progression of EH for the LSL-K-rasG12D;Rac1flox/+ mice are shown at 6, 12, and 24 wks (A, B, and C, respectively). The epithelial cells grow into the lumen of the airways, eventually obstructing the entire lumen (C). Similar lesions were found in the LSL-K-rasG12D; Rac1flox/flox mice; however, the appearance and progression of EH was delayed by 6 wks in this group.

In addition to assessment of tumor initiation, we examined the ratio of tumor volume to lung volume (T/L ratio) at the various time points. In the LSL-K-rasG12D;Rac1flox/+ mice the T/L ratio was at 1.23% at 6 weeks, 4.82% at 12 weeks, and rose to ∼16.4% and 26.46% at the 18- and 24-week time points, respectively. In comparison, T/L ratios in the LSL-K-rasG12D;Rac1flox/flox mice were greatly reduced. At the 6-week time point, the tumor-to-lung volume ratio was at 0.08%. At 12 weeks postinfection, the T/L ratio was 0.68%. It was 3.5% and 9.54% at the 18- and 24-week time points, respectively (
Figs. 1A and
2).

To assess whether the loss of Rac1 resulted in reduced tumor initiation and/or slower progression of the tumors that did arise, we compared the distribution of tumor sizes between the two groups at the various time points. As shown in
Fig. 1D, a statistically significant difference in tumor size was observed between the groups at all time points examined. Based on tumor genotyping data (see below), we believe this difference is due to an indirect effect of incomplete recombination of the conditional Rac1 allele in emerging tumor clones.

Finally, animals of both genotypes were infected with Ad-Cre at 8 weeks of age and were kept until they required sacrifice due to their tumor burden. The LSL-K-rasG12D;Rac1flox/+ mice lived an average of ∼240 days following infection, and the majority of mice in this group succumbed between 205 and 255 days after infection (
Fig. 1C). The LSL-K-rasG12D;Rac1flox/flox mice survived to an average of 320 days postinfection, with the majority of animals dying between 275 and 325 days (
Fig. 1C). In all cases, the animals had a high lung tumor burden at sacrifice.

Histologic examination of the tumors and precancerous lesions that arose in the different groups indicates that they were grossly similar but arose at different times. In both groups, three distinct types of lesions were found. Atypical adenomatous hyperplasia (AAH) was present at 6-weeks postinfection in both groups of mice, although at much higher levels in LSL-K-rasG12D;Rac1flox/+ mice (
Fig. 2). Small papillary adenomas were evident at 6-weeks postinfection in the LSL-K-rasG12D;Rac1flox/+ mice and larger adenomas at 12 weeks; adenocarcinomas were present at 18 and 24 weeks. In comparison, adenomas were not readily apparent in the LSL-K-rasG12D;Rac1flox/flox mice until the 12-week time point, and adenocarcinomas were observed rarely at the 18-week time point and became more evident at the 24-week time point (
Fig. 2). Both groups of mice also presented with epithelial hyperplasia (EH) of the bronchiole. As previously reported, a large number of the AAH and adenomas were continuous with the EH (
15). EH lesions were clearly evident in the LSL-K-rasG12D;Rac1flox/+ mice at 6 weeks postinfection and, at later time points, had grown to fully obstruct some of the bronchioles (
Fig. 2A–C). In comparison, in the LSL-K-rasG12D;Rac1flox/flox mice, EH lesions were only evident at the 12- and 18-week time points; at 24 weeks, a small number of lesions obstructed bronchioles.

To assess the status of the K-ras and Rac1 alleles in the tumors, we microdissected several tumors from the various groups at the 12- and 18-week time points, and the status of the alleles was evaluated by PCR. As shown in
Fig. 3
, in all tumors, the LSL-K-rasG12D allele had undergone Cre-mediated removal of the stop element. Likewise, the Rac1 allele was recombined efficiently in the tumors isolated from the LSL-K-rasG12D;Rac1flox/+ mice. However, in all samples from tumors from the LSL-K-rasG12D;Rac1flox/flox mice, a band representing the unrecombined allele of Rac1 was still evident, reflecting incomplete inactivation of Rac1. This was not due to the contamination of the tumor samples by nontumor tissue, as indicated by the absence of non-recombined LSL-K-rasG12D allele in the same samples [
Fig. 3A; compare control lane (contam) to tumors]. Furthermore, we confirmed the expression of Rac1 in the tumors from the LSL-K-rasG12D;Rac1flox/flox mice group by immunohistochemistry. In agreement with the PCR data, we were able to detect Rac1 expression in all tumors examined (
Fig. 3B). Thus, lung tumors initiated by oncogenic K-ras are dependent on Rac1 function, and only cells that escape inactivation of both alleles of Rac1 are able to progress efficiently in tumorigenesis. This result may help explain the difference in rates of tumor progression in control versus LSL-K-rasG12D;Rac1flox/flox mice. If within a clone of K-ras–induced cells most but not all of the cells undergo complete Rac1 recombination, the development of subsequent tumors would be expected to be slower than control tumors with uniform Rac1 activity.

Analysis of the K-ras and Rac1 allele status in tumors. Tumors were excised from the LSL-K-rasG12D;Rac1flox/+ mice at 12-wk (lanes 1–4) and LSL-K-rasG12D;Rac1flox/flox mice at 18-wk (lanes 5–8) time points by microdissection. A, status of the K-Ras allele. B, status of Rac1 alleles. The alleles were assessed by PCR on genomic DNA extracted from these tumors. The presence of contaminating nontumor tissue was evaluated through the detection of the non-recombined LSL-K-rasG12D allele (Contam Lane control). Only tumors that had no detectable contamination were assessed for the status of the Rac1 allele. The data in this figure are a representation of more than 100 tumors from six different mice in both the LSL-K-rasG12D;Rac1flox/flox and LSL-K-rasG12;Rac1flox/+ groups. C, staining of sections of lungs from LSL-K-rasG12D;Rac1flox/flox mice with an anti-Rac1 antibody. Sections from four different mice from the 6-, 12-, 18-, and 24-wk time points were examined; magnification, ×4. In all cases, the expression of Rac1 was detected in all tumors.

A trivial explanation for the results presented above is that absence of Rac1 function leads to cell lethality, thereby preventing the outgrowth of any cell in which complete Rac1 deletion occurred. Because of the difficulty in assessing cell lethality in the lung, we turned to primary epithelial cells derived from the kidney. Specifically, BMK cells were isolated from newborn pups of the LSL-K-rasG12D, LSL-K-rasG12D;Rac1flox/flox and Rac1flox/flox genotypes described above. Because the tumors that arise in LSL-K-rasG12D mice are epithelial in origin, BMKs are an appropriate system in which to assess the cellular effects of K-ras activation and Rac1 deletion. BMK cells were infected with Ad-Cre and were examined 72 h later for rearrangement of the various alleles by PCR (
Fig. 4B
and Supplementary Fig. S1). Cell populations displaying between 90% and 100% recombination were further examined to determine the effect of loss of Rac1 (either alone or in the context of K-ras activation) on cell proliferation and viability. BMK cells infected with empty adenovirus served as controls.

A, proliferation of BMK cells from the LSL-K-rasG12D, Rac1flox/flox, and LSL-K-rasG12D;Rac1flox/flox mice. BMK cells were treated with control Ad-empty or Ad-Cre, and 72 h after infection, cell cycle characteristics were determined by flow cytometry and PI staining. The data shown are representative of three independently derived cultures from each genotype. B, status of the various alleles in the BMKs was verified by PCR. Only cells displaying 90% to 100% recombination of both alleles were used in the described experiments. Gel images are cropped, and full-length gels are presented in Supplemental Fig. S1. C, proliferation of BMKs with the different allele combinations. BMK cells were treated with Ad-Cre, plated, and counted daily. All points are averages of triplicate samples, and the data shown are representative of three independently derived cultures from each genotype. The difference in cell number between BMKs of the Rac1flox/flox and LSL-K-rasG12D;Rac1flox/flox genotypes is statistically significant at both time points postinfection. Bars, SD, P < 0.001, standard t test.

Flow-cytometric analysis was used to examine the cell cycle characteristics of BMKs from each of the three genotypes (
Fig. 4A), and the data shown are representative of three independently derived cultures from each genotype. LSL-K-rasG12D BMKs infected with Ad-Cre displayed a higher percentage of cells in G1 and S phases (40.8% and 15.2%, respectively) than control cells (33.4% and 11.5%, respectively). We believe that this is indicative of an increased rate of exit from G2-M and increased cellular proliferation, as supported by data obtained from daily cell counts in LSL-K-rasG12D BMKs (see
Fig. 4C and below). Importantly, Ad-Cre–infected Rac1flox/flox BMKs displayed similar growth kinetics to LSL-K-rasG12D BMKs (39.6% of cells in the G1 phase and 13.5% in the S phase compared with controls at 34.9% in the G1 phase and 11.6% in the S phase). Thus, in BMK cells, loss of Rac1 function alone is not lethal and is compatible with proliferation. In contrast, following Ad-Cre infection, LSL-K-rasG12D;Rac1flox/flox BMKs displayed a decrease in the percentage of cells in the G1 phase (27.1% compared with 35.8% in control cells) and an increase in the percentage of cells in G2-M (56.4% compared with 50.4% in control cells), indicating a decreased rate of exit from G2-M and decreased cellular proliferation (
Fig. 4A).

These data were further corroborated by experiments in which BMK cells of the different genotypes were counted daily to assess cell proliferation (data illustrated are representative of three independently derived cultures from each genotype). These studies showed that the LSL-K-rasG12D;Rac1flox/flox BMKs proliferated significantly slower than both LSL-K-rasG12D and Rac1flox/flox BMKs, as indicated by significantly decreased cell numbers at 3 days postinfection with Ad-Cre (
Fig. 4C). However, although the absolute cell numbers of the LSL-K-rasG12D;Rac1flox/flox BMKs were still lower at day 4 postinfection, their actual rate of proliferation between day 3 and day 4 was comparable to the LSL-K-rasG12D and Rac1flox/flox BMKs. This may have been due to the selection of LSL-K-rasG12D;Rac1flox/flox BMKs that had escaped complete inactivation of both alleles of Rac1. Unfortunately, because of the induction of cellular senescence in culture at later time points, we were not able to continue the cell counting experiments beyond 96 h postinfection. However, the data show that there is a specific requirement for Rac1 function in primary epithelial cells expressing oncogenic K-ras. This synthetic requirement for Rac1 function may explain the inhibitory effects of Rac1 deletion on lung tumor formation as described above.

Increasing evidence has implicated the small GTP-binding proteins of the Rho family as important factors in Ras-induced transformation. Our data show a requirement for Rac1 in K-ras–induced transformation in vivo. We have shown that no tumors develop in the absence of Rac1 in a K-ras–induced lung tumor model. Similarly, recent work has shown a requirement for the Rac-GEF, Tiam1, in DMBA-induced skin tumors (
10). The Tiam−/− mice show reduced levels of activated Rac and were resistant to H-Ras–induced skin tumors (
10). Interestingly, K-Ras has been shown to be a more effective activator of Rac1 than H-Ras (
19). The BMK data presented here show that Rac1 is not essential for cellular proliferation, but rather, the requirement for Rac1 is specific in the context of K-Ras activation. These findings are consistent with previous work demonstrating a requirement for Rac1 in Ras-induced foci formation (
7,
8).

In vitro studies have suggested that Rac1 functions to suppress the apoptotic pathway, which is activated by activated Ras. The ectopic expression of Rac1 has been shown to inhibit apoptosis of tumor cell lines and transformed fibroblasts (
20). This function of Rac is thought to be mediated via the activation of NF-κB (
21) and/or by the phosphorylation of targets, such as the anti-apoptotic protein BAD, via the p21-activated kinase 1 (
22,
23). It is also possible that the effect of Rac1 is mediated through the requirement for Rac1 signaling in the up-regulation of cyclin D1, which is crucial for Ras-induced tumorigenesis (
24). This aspect of Rac1 function could be mediated via the activation of c-jun-NH2-kinase and/or NF-κB, which both control the transcription of cyclin D1 (
25,
26). Additional evidence indicates that Rac1 could also function to regulate cyclin D1 via the extracellular signal-regulated kinase (ERK) pathway because the p21-activated kinases have been shown to directly phosphorylate Raf-1 and mitogen-activated protein/ERK kinase 1 (
27,
28). Future studies employing effector domain mutation in Rac1 will be instrumental in elucidating the downstream pathways required for Ras transformation.

Finally, the finding that Rac1 is required specifically in the context of activated K-Ras raises the possibility that targeting Rac1 in Ras mutated tumors would be therapeutically beneficial. Although this is an attractive possibility, the long-term effects of Rac inhibition may be problematic. For example, although the loss of Tiam1 function initially confers a resistance to H-Ras–induced skin tumors, it may play a role in tumor progression later in the tumorigenic process (
10). Identifying the pathways downstream of Ras activation and the points of interaction between them are crucial for the development of an integrated map of cellular signaling networks. The use of animal model systems, in combination with in vitro systems, will allow exploration of signaling pathways under physiologically relevant conditions.

Acknowledgments

Grant support: Cancer Center Support (core) grant P30-CA14051 from the National Cancer Institute.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Erica Jackson and Julien Sage for helpful discussions and comments. We thank Denise Crowley for histologic preparation and Karen Kineman for technical assistance. T. Jacks is an investigator of the Howard Hughes Medical Institute.